The human body depends on oxygen in order to live. In fact, virtually all life on our planet requires oxygen. Unfortunately, when oxygen is used intracellularly to fuel the aerobic chemical processes required for life, toxic oxidative by-products, referred to as reactive oxidative species (ROS) are left behind in the cell.
The body has three major enzymes which detoxify ROS. These enzymes are superoxide dismutase (SOD), which catalyzes the transformation of the superoxide anion to peroxide; catalase, which catalyzes the transformation of hydrogen peroxide into water; and glutathione peroxidase ("GP"), which catalyzes the destruction of hydrogen and lipid peroxides, using glutathione as an electron donor. GP occurs in various forms, the most significant of which are plasma GP ("GPP") and erythrocyte GP ("GPE"). Of the three antioxidant enzymes, SOD and GP are the most important.
The presence and the activity of these enzymes play a critical role in human health. Primarily, the balance between the components of the antioxidant defenses appears to be of prime importance for the cellular resistance to what is called "oxidative stress", that is, the intracellular imbalance between prooxidants causing release of ROS during metabolism and antioxidants which destroy the ROS.
The invention has several embodiments. Broadly considered, the invention deals with the control, or the treatment of stress, and under certain circumstances described herein, the alleviation of the symptoms of stress. The invention in one of its very interesting applications addresses the universal problem of mortal animals and human beings, the problem of aging. For this purpose, the invention provides a transgenic non-human animal which produces or overproduces SOD and/or GP. These transgenic animals are an ideal, useful model to evaluate and/or monitor in a practical manner the effect of SOD and/or GP when these transgene animals are subjected to various stressful conditions, such as the diseases discussed herein and/or on the aging phenomena.
Several theories have been postulated to explain why cells age and ultimately die. Until relatively recently, many scientists subscribed to the theory of a genetic basis for senescence. Since the removal of the elders from a population would reduce the drain on resources and so allow the younger individuals freer access to those resources, it was felt that aging was programmed in genes whose sole function is to cause senescence in the organism. This theory has lost favor, however, because wild animals usually do not survive long enough in the wild to become senescent. Additionally, until recently, human life expectancy was not the 75 years experienced in today's industrial societies, but was less than 40 years.
Today, it is widely believed that aging results from repeated minute damage to cells which accumulates over time, causing loss of cellular function. This damage is due to ROS released as a result of the use of oxygen for energy metabolism.
According to this theory, ROS made up of free radicals and highly oxidative compounds are generated during metabolism.
These ROS, such as superoxide (O.sub.2.sup.-), peroxide (H.sub.2 O.sub.2), and hydroxyl radical (OH.sup.-), are highly toxic and cause serious damage to various cellular components, such as chromatin, structural macromolecules, cellular and organelle membranes, and other components. Therefore, in order to protect itself, organisms have developed defense mechanisms to remove these toxic ROS.
Several diseases in humans have been associated with an imbalance of the antioxidant defense system with an increased or decreased SOD expression relative to GP expression. Examples of such diseases include Alzheimer's Disease, Down's Syndrome, amyotrophic lateral sclerosis, and Parkinson's Disease.
There is convincing evidence that prolonged oxidative stress plays an important role in carcinogenesis. It is believed that ROS cause mutations in DNA which may ultimately lead to cancer.
Results of several studies indicate that an increase or a decrease in activity of one of the antioxidant enzymes without concomitant change in activity of the other may lead to the prooxidant state within the cell and could contribute to the aging phenomenon and to various disease processes. For example, an increase in SOD activity would lead to the increased production of peroxide, a potent prooxidant. Since GP acts to detoxify peroxide, inadequate supplies of GP to deal with the increased peroxide load due to increased SOD activity, can cause an increase in cellular damage.
It is evident that an imbalance of the detoxifying enzymes has adverse effects on the mammal and that it will be very desirable to control and properly balance the role and effect of the principal antioxidant enzymes.
In addition, the modes of action of several toxic substances involve the production, overproduction or depletion of reactive oxidative species. For example, benzidine, a pollutant present in automobile emissions, has been shown to cause increased levels of ROS. Another poison, benzene, a common industrial pollutant, causes increased production of ROS in vitro. The increase in ROS associated with benzene is believed to be the cause of benzene's toxicity. A third example is that of acetaminophen, a harmless and useful analgesic when taken at therapeutic levels. At toxic levels, however, acetaminophen causes depletion of GP from the liver, resulting in liver damage.
An intriguing aspect of the role of antioxidants is the sensitivity of cells to hyperthermia, often leading to death of the cell. In hyperthermic cells, levels of hydrogen peroxide build up rapidly due to the increased rate of metabolism due to the increased temperature. High levels of hydrogen peroxide are thought to function as a signal to the cell to make heat protective proteins. Tumor cells have increased levels of GP and are more sensitive to hyperthermia than are normal cells. It is believed that the high levels of GP in tumor cells leads to depletion of hydrogen peroxide so that heat protective proteins are not made, thus rendering the cell more sensitive to hyperthermia.
It is evident from this review that the literature reports often contradicting or inconclusive results that do not provide guidance as to how to control ROS species in mammals in a reliable and reproducible manner. Further, the literature provides often contradicting and inconclusive results of the effects or changes in the production of ROS in mammals.
Significantly, no report has been found of a double transgenic animal bearing the genes for both SOD and GP. Such a transgenic animal is an ideal practical model to determine the effects of disruptions of the balance between the two enzymes due to time, environmental conditions and/or various agents, a balance that has been shown to be crucial in preventing cell damage due to ROS. The double transfgenic animal would also contribute to the elucidation of many of the mysteries of aging.